Feature Articles

Protein Analysis Technologies Overview

A Host of New Product Introductions Highlighted at PEGS Invigorates the Field

The QCMA1 system, which was launched earlier this year by Sierra Sensors, was one of many products on display at CHI’s “Protein Engineering Summit (PEGS).” QCMA1 is an automated, dual-sensor, label-free protein-analysis system capable of determining binding specificity, kinetics, affinity constants, concentration, thermodynamics, and structural changes, according to Christopher Whalen, CEO at Sierra Sensors. The system features quartz crystal microbalance detection technology and a continuous flow sample delivery system.

Wasatch Microfluidics also displayed its Continuous Flow Microspotter™ (CFM), designed for uniform, reproducible deposition of biomolecules on an array surface for kinetic and quantitative analyses. The 48-spot CFM printhead is composed of a 3-D network of microchannels that can simultaneously deliver 48 samples in a 4 x 12 configuration to 400 micron square spots.

Solution containing the sample of interest (such as a protein mixture or cells), reagent, or wash buffer flows continuously through the inlet and outlet wells and over the substrate. Solution can cycle bidirectionally until the desired deposition level is achieved. This is particularly advantageous for capturing low abundance proteins, noted Steve Nyikos, Ph.D., scientist at Wasatch Microfluidics.

Compared to a conventional pin printer, the CFM system offers enhanced spot uniformity at low protein concentrations and eliminates spreading or “comet tailing” at higher concentrations, according to the company. It is compatible for use with crude lysates, “eliminating the need to purify a sample,” said Dr. Nyikos. For example, the system can be used to print antibodies directly from a crude sample onto a capture surface in sufficient quantities to enable label-free SPR analysis. Additionally, it allows users to perform a wash step to remove unadsorbed protein while the printhead is in place.

EMD Biosciences, through a licensing agreement with Invitrogen, introduced Gateway® Nova DEST™ Vectors. These expression vectors combine the advantages of pET-inducible protein expression and control elements with the Gateway cloning technology, which allows construction of various expression clones for the same target gene by creating an entry clone and then performing in vitro, site-specific recombination between the entry clone and the destination vectors.

Offering a selection of N- and C-terminal fusion tags, the new DEST vectors include GST-Tag™, Hi-Tag®, S-Tag™, Strep-Tag® II fusions for affinity purification, and Nus-Tag™ and Trx-Tag™ to enhance protein solubility, as well as pCOLA-DEST™ vectors for co-expression of multiple targets in E. coli.

Going Label-Free

FortéBIO introduced the Octet RED System, a benchtop instrument based on BioLayer Interferometry and designed for label-free biomolecular kinetics analysis of protein/small molecule interactions. Joy Concepcion, product manager at FortéBIO, explained that the system can operate in two modes—kinetics characterization or quantitation of expressed protein—without flow-cell fluidics.

For higher-throughput interaction analysis, it runs eight samples in parallel and utilizes disposable, single-use optical biosensors. Test volumes range from 100–200 µL. Proteins are immobilized or captured on the biosensor surface with covalent coupling of proteins containing a primary amine to an amine reactive biosensor; a streptavidin biosensor surface chemistry binds biotinylated proteins or precoated biosensors such as protein A. For kinetic screening, the system can process 96 samples in two hours; quantitation of 96 protein samples takes about 30 minutes.

GWC Technologies recently began offering label-free array analysis of molecular interactions as a service on its SPRimager®II platform. Quantitative changes in reflectivity demonstrate real-time binding of a substrate to targets immobilized on an SPR chip. Laboratories using the SPRimagerII can purchase spot-ready chips composed of a gold surface on glass that can be modified to suit particular target types and offer flexible array formats. SpotReady™ chips can accommodate 16 or 25 spots, applied using a manual pipette. Use of the SPRchip™ and a spotting robot enables placement of at least 400 spots on the array surface, according to Voula Kodoyianni, Ph.D., CSO.

One advantage of this open platform design, she added, is the ability to “compare different spotting chemistries side-by-side,” for example, antibodies may behave differently depending on the chemistry used to immobilize them on the chip surface.

Another advantage is the ability to expose the whole array to the same analyte in a 1 cm flow cell, which “eliminates the channel-to-channel variability inherent in multichannel systems and allows for simultaneous capture of data for all probes on the array,” according to the company. “Users can sequentially expose the array to multiple analytes/antigens,” said Dr. Kodoyianni, facilitating analysis of ligand-dependent binding and competitive binding interactions.

Also exhibiting at “PEGS” was Attana. The firm developed the A100 system for label-free, real-time, continuous-flow detection of molecular interaction studies to characterize antibodies and other proteins. The instrument’s biosensors, based on QCM technology, come in a variety of sensor surfaces including a polystyrene surface that can be used to obtain real-time kinetic, affinity, and specificity data for a target immobilized on the sensor surface and exposed to an interacting molecule.

Attana touted the availability of a variety of application notes that demonstrate the applicability of the A100 system for affinity and kinetic determination of antibody-antigen interactions, selection and characterization of mAbs for use in sandwich assays, and on-line studies of protein-cell interactions.

Protein Design

Codon Devices described Constructive Biology™ as an enabling technology for engineering biological products such as genes, proteins, metabolic pathways, and cell lines. The company’s BioFAB™ DNA-assembly platform produces libraries of defined DNA sequences in lengths from 1,000 to upwards of 35,000 base pairs.

Dasa Lipovsek, Ph.D., director of protein engineering, described the company’s new BioLOGIC™ protein engineering platform, which is based on the ability of BioFAB to design and construct synthetic DNA genes accurately and inexpensively.

“We can make a large DNA library with defined sequences that accurately reflect the design rules,” said Dr. Lipovsek. Utilizing computational, modeling, and analytical tools, BioFAB generates sets of thousands to a hundred thousand predictive sequences.

In contrast to approaches based on random mutagenesis, Codon Devices employs a directed library design method to produce custom-defined sequences that incorporate mutations at specified rates and positions, she added. DNA error filtration using immobilized MutS to detect mismatched base pairs leads to removal of the faulty double stranded DNA sequence from further assembly.

BioFAB is at the core of the BioLOGIC process, which also relies on structure-based computational protein design tools to generate 3-D protein models capable of depicting molecular interactions at an atomic level. Algorithms then predict the amino acid changes intended to result in a function of interest.

Once a library of predictive genes is produced and cloned into a host, the company uses a cell-surface display platform for high-throughput selection of the proteins of interest. Each protein remains associated with its corresponding DNA sequence, so when we “select a protein, the gene comes along for the ride,” said Dr. Lipovsek.

She described how the company’s secretion-and-capture selection method enables the yeast cell to secrete the protein, which adheres, by means of a sticky label, to the outside of the cell, with the plasmid/oligonucleotide remaining inside the cell. By exposing the protein-studded yeast to a target antigen one can characterize the proteins for binding affinity, activity, and stability, select the proteins of interest, and extract and sequence the DNA. Next, the selected gene is used to express sufficient protein for further characterization.

“We then put the oligo into a new host, express the protein,” and produce enough for biochemical characterization studies, she explained.

In addition to ongoing internal projects, Codon Devices is involved in collaborative efforts focusing on therapeutic protein discovery and lead optimization as well as biofuels development.

Designed for high-throughput screening to identify the optimal conditions for refolding of a particular protein, EMD Novagen iFOLD™ systems allow researchers to perform protein refolding screens in a 96-well format. Achieving high yield of recombinant proteins in E. coli-based expression systems is relatively straightforward and economical, but producing properly folded proteins required for functional and structural studies is often challenging, according to Alla Zilberman, Ph.D., director of product management at Novagen.

A substantial amount of the desired protein output may be present in inclusion bodies, which are dense, insoluble aggregates of misfolded proteins. These can be solubilized and purified, “but defining conditions that promote refolding of a chemically denatured protein into its native conformation is empirical and often time-consuming,” noted Dr. Zilberman. iFOLD technology allows for systematic evaluation in parallel of a large number of refolding conditions, she added.

EMD launched its iFOLD System 3, which assays 96 different refolding conditions and incorporates Triton-X detergent to wash the inclusion bodies and novel FoldACE™ additives that enhance protein refolding and stabilize the refolded proteins. The company’s iFOLD System 2 product is a detergent-free screening system.

At the meeting, Sidec elaborated on protein tomography, which provides 3-D visualization of individual antibody molecules bound to a target, according to Michael Smith, Ph.D., field scientist. The key feature of the technology, Dr. Smith explained, is single molecule detection and visualization, which enables both epitope mapping and mechanism-of-action studies. The research is able to determine antibody-binding sites by direct inspection of the binding event in three dimensions, and to detect conformational changes of individual target molecules in the presence of an agonist or antagonist, allowing the study of how binding affects target function. With a resolution of 25-30 Å, protein tomography can be used with purified protein and protein complexes or to view where and how tagged antibodies bind to their targets inside cells.

“We work with our clients to develop a better IP strategy and an improved product positioning for therapeutic antibodies through improved drug candidate selection, lead classification, and target mechanism studies,” said Dr. Smith.

The second poster highlighted the advantages of Promega’s wheat germ-based TNT® SP6 cell-free protein expression system for difficult-to-express proteins. The single-tube system can achieve protein yields as high as 400 µg/mL in batch mode. Promega has demonstrated efficient expression using the TNT SP6 system of several human proteins that could not be expressed as soluble proteins in E. coli using a variety of N-terminal solubility tags, it reported.

RedSee™ is a fusion protein that, when properly folded and linked to a recombinant protein, produces a red color that is detectable by the human eye in culture and during protein purification. Blue Sky Biotech developed the RedSee technology for visual tracking and optimization of recombinant protein expression and purification. A poster describing the purification and activity assessment of RedSee-tagged JNK3 and TEV proteases showed that the tagged proteins retain their activity when fused to RedSee and after proteolytic removal of the tag. Blue Sky presented preliminary data suggesting that the RedSee tag may confer improved solubility to fusion partners, even when used in combination with affinity tags.

Northstar BioProducts introduced two new kits for release of O-linked glycans, the Ludger Liberate™ hydrazinolysis and Orela kits. Developed as a tool for biopharmaceutical glycoprofiling during drug development and for glycan characterization to support regulatory submission, the hydrazinolysis kit allows for release of both N- and O-linked glycans and creates free reducing termini suitable for fluorescent labeling. The Orela kit is suitable for high-throughput, routine analysis of O-glycosylation by quality control labs.

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